Bacteria in the class Mollicutes are parasitic organisms commonly contaminating eukaryotic cell culture systems. Members of the Mollicutes are among the smallest and simplest of the prokaryotes. Because of their small size and lack of a rigid cell wall, Mollicutes pass easily through filters intended to remove bacterial contaminants. Mollicutes contamination of a cell culture is problematic because it can negatively impact cell growth, alter metabolism and lead to unsafe final products. Mollicutes compete with cultured cells for essential nutrients and produce toxins that can cause cell death, all of which can impact the quality and productivity of cell cultures. These organisms are therefore of significant concern to the biopharmaceutical industry, which is dependent on continuous cell culture for the production of drugs, vaccines, and other biologics.
Mollicutes include species from the genera Mycoplasma, Acholeplasma and Spiroplasma. Mollicutes can originate from mammalian, avian, insect, plant or fish cells. These organisms also have limited biosynthetic capabilities, making them dependent on external sources for essential nutrients and cofactors. Many species of Mollicutes have therefore evolved to become intracellular parasites. Thus, contaminating Mollicutes are present extracellularly and intracellulary in a host-cell environment.
Current culture methods for detection of Mollicutes are limited by the time required for the growth of these fastidious organisms. As a result, detection of Mollicutes using these methods can take 28 days or longer, a timeframe that is not compatible with today's fast pace of pharmaceutical manufacturing and distribution. Access to rapid, simple and relatively inexpensive methods for the detection of Mollicutes would enable the routine testing and proactive quality control of cell cultures, raw materials, equipment, fixtures and the like.
Compared to cell-culture methods, nucleic acid amplification tests are much more rapid detection methods. However, there is currently not a satisfactory nucleic acid amplification test available for detecting Mollicutes contamination of a cell-culture. Currently available nucleic acid amplification tests suffer from problems with identifying more than one or a few species of Mollicutes per assay. Further, for the assays that are designed to identify more than one species of Mollicutes, there are commonly problems with cross reactivity to related bacteria or nucleic acids that may be present in the sample. Further still, the currently available tests fail to detect some of the Mollicutes species known to contaminate and negatively impact cell cultures. Thus, there is a need in the art for an improved nucleic acid amplification test for identifying Mollicutes contamination.